Nucleotide excision repair (NER) protects human cells by removing harmful DNA adducts formed by environmental toxins and solar UV irradiation. Defects in NER in humans lead to the DNA repair disorder xeroderma pigmentosum, which is characterized by high predisposition to skin cancer, neurological abnormalities and premature death. The repair of damaged DNA by NER also has a downside ? it contributes significantly to the development of resistance of tumors to treatment with antitumor agents, in particular cis- and carboplatin, two of the most widely prescribed drugs in oncology. We propose to leverage the combined expertise of the Scharer and Chazin laboratories in chemistry, biochemistry, cell biology, structural biology, and small molecule discovery to elucidate how the scaffold protein XPA coordinates the assembly and organization of NER incision complexes. Despite its modest size (273 residues), XPA functions as the central scaffold of NER complexes, interacting with 4 key NER proteins as well as DNA. However, how XPA is recruited to the site of damage and positions other factors through its various interactions remains poorly understood. Moreover, given its essential role in organizing and orchestrating the trajectory of NER complexes, XPA is an attractive potential Achilles Heel to target for suppressing NER. Aim 1 will test the hypothesis that TFIIH recruits XPA to sites of UV damage through a proposed interaction interface involving the C-terminal region of XPA and the p8 subunit of TFIIH. We will biochemically and structurally characterize this interaction and determine how mutations in the interface that disturb this interaction affect NER in vitro and in vivo. Abolishing the interaction between XPA and TFIIH will also address the long-standing question of whether the steps following damage recognition are the same for global genome and transcription-coupled NER ? the arrival of TFIIH and XPA is the first step common to both pathways. The action of TFIIH on damaged DNA creates an open ?NER bubble? that provides a landing platform for XPA and RPA and is required for NER incision. The scaffolding function of XPA is reliant on its coordination with RPA, which binds the undamaged strand. Aim 2 will determine the molecular basis and functional implications of the coordinated action of XPA and RPA. Structural, biochemical, and biophysical approaches combined with cellular assays will test the hypothesis that the two interaction sites between XPA and RPA are simultaneously engaged and contribute to NER in a cooperative fashion. Based on these results, our expertise in fragment based molecular discovery will be used to develop and validate initial inhibitors targeting XPA-RPA interfaces. These studies are expected to provide: (i) dramatic new mechanistic insights into the central role of XPA in assembling and coordinating the NER machinery; (ii) the identity of XPA interaction surfaces that are critical to NER; (iii) proof of principle that interfaces between XPA and other NER factors are suitable targets for evaluating the potential of overcoming tumor resistance to DNA damaging therapies by suppressing NER.